Clear-cell renal carcinoma (RCC) is the predominant form of renal carcinoma. Medical treatment of RCC is generally ineffective with a median life span of only 1 year in patients with metastatic disease. RCC is derived from mutations in the von Hippel-Lindau (VHL) gene. In VHL-deficient cells, the hypoxia inducible transcription factor, HIF-2 alpha, is stabilized and up-regulates several genes that support tumor growth. Down-regulation of HIF-2 alpha is necessary and sufficient to block tumor formation in animal models. Reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide are involved in the signaling pathways mediating many stress and growth responses. NAD(P)H oxidase(s) are a major source of ROS implicated in renal oxidative stress. We have strong evidence that VHL-deficiency plays a critical role in the pathogenesis of RCC through increased generation of ROS by the NAD(P)H-dependent oxidase subunits, Nox4 and p22phox, which are critical in maintaining HIF-2 alpha protein expression in the absence of VHL. The goal of our studies is to elucidate the molecular mechanisms linking VHL-deficiency, increased expression and or activation of NAD(P)H oxidases and increased expression of HIF-2 alpha and target genes involved in the development and progression of RCC in vitro and in vivo. AIM1 will determine the mechanisms by which VHL regulates the catalytic unit of the NAD(P)H oxidase Nox4 and its membrane binding partner, p22phox. VHL is part of an E3 ubiquitin ligase complex that poly-ubiquitinates specific proteins for regulated protein degradation through the 26S proteasome pathway. This process requires the binding of VHL to the substrate. It will be determined if Nox4 or p22phox protein are stabilized in the presence of proteasome inhibitors and if Nox4 and p22phox are post translationally ubiquitinated in vitro and in vivo in a VHL-dependent manner. In vitro and in vivo binding studies will be performed to determine if VHL binds Nox4 and p22phox. Alternatively, Nox4 may be up- regulated in VHL-deficient cells through enhanced transcription of the mRNAs. We will determine if Nox4 mRNA levels are increased in VHL-deficient cells by Northern Blotting and quantitative real time PCR. Nox4 promoter activity will also be examined to explore a putative feed back loop of HIF-21 on the Nox4 promoter as a potential mechanism of Nox4 expression. The second AIM of this proposal will elucidate the mechanisms by which the Nox oxidase components maintain HIF-2 alpha protein expression in VHL-deficient cells. We will specifically determine if the maintenance of HIF-2 alpha by ROS in VHL-deficient cells requires on-going transcription, mRNA stabilization or activation of signal transduction pathways that mediate translation. The third AIM will examine the effect of stable silencing of Nox4 and p22phox or superoxide antioxidants on HIF-2a transcriptional activity, tumor growth and cell invasiveness (other characteristics associated with VHL- deficiency) in vitro and in vivo. PUBLIC HEALTH RELEVANCE: VHL-deficiency plays a critical role in the pathogenesis renal clear cell carcinoma (RCC). The goal of this project is to elucidate pathogenic mechanisms of development and progression of VHL-deficient renal cell carcinoma, through increased generation of reactive oxygen species by the NAD(P)H-dependent oxidases which are essential in maintaining HIF-21 protein expression, a critical factor necessary and sufficient for the progression of VHL-deficient tumorigenesis. This project may result in the development of novel and specific therapeutic regiments to treat RCC.